1. Field of the Invention
The detection and identification of molecules is the aim of the present application. In particular, the invention is directed to the labeling and detection of amine-functionalized biomolecules such as amino acids, oligonucleotides, carbohydrates, peptides and peptide nucleic acids with detectable marker molecules such as biotin or fluorescein by post-synthetic derivatization with marker molecule esters of 1-Phenylpyrazolin-5-one (XPP).
2. Description of the Prior Art
The need for detecting chemicals and molecules is an ongoing one in the biotechnology industry. It is a well known fact that biochemical assays and procedures in general are dependent upon the accurate identification and quantification of biomolecules, including pharmaceuticals, proteins, peptides, DNA, RNA, and PNA (Peptide Nucleic Acid). Many of these biomolecules are directly detectable by virtue of their intrinsic properties, and devices have been built to measure their ultraviolet excitation, infrared absorption, mass spectra, atomic absorption, fluorescence emission, nuclear magnetic resonance, and a host of other identifying data. However, certain classes of molecules are difficult to directly detect, and these are "labelled" with a marker molecule that is itself directly detectable.
Biomolecules are a particular class of difficult-to-detect molecules. Often they do not absorb in the ultraviolet region because of their lack of extended aromatic structures. Generally, they are not analyzable by mass spectrometry because they are too big to be rendered both ionic and volatile, and the technique requires very expensive and complex instrumentation. Infrared spectra are useful only for simple molecules. In addition, the amounts of DNA, RNA, PNA, amino acids and peptides that are present in today's sensitive recombinant assays are so low that new labels and attachment chemistries are constantly being developed in an effort to lower detection limits. Today, radioactive labels such as .sup.32 P are used in the 5' labeling of oligonucleotides, but they have unstable shelf lives, and the attendant hazards of radiation present significant drawbacks. Thus, affinity labels such as biotin were adopted as non-radioactive labels. Biotin is strongly bound by the proteins avidin and streptavidin, which in turn may be conjugated to an enzyme such as alkaline phosphatase, or equivalently horseradish peroxidase. Addition of the avidin/streptavidin-enzyme complex to a biotin-containing target allows detection by the addition of a color indicator, which is facilitated by enzymatic reaction. In addition, biotinylated biomolecules may be extracted from a solution containing other compounds by adsorption to a streptavidin affinity column. Other non-radioactive labels include fluorescent detection by excitation of a biomolecule-fluorescer complex. Fluorescent labels such as fluorescein, coumarin, calcein, ethidium homodimer and intercalation dyes in general are common.
A variety of methods have been developed for the addition of biotin to oligonucleotides. See Wilchek, M, et al., Anal. Biochem. 171: 1-32 (1988). Chemical addition of biotin to a completed oligonucleotide is usually performed in a two-step procedure involving synthesis of an oligonucleotide modified by a primary amino group, followed by coupling, in aqueous solution, to a biotin N-hydroxysuccinimide ester. Chollet, A., et al., Nucleic Acids Res. 13:1529-41 (1985); Sinha, et al., Nucleic Acids Res. 16:2659 (1988); Bengstrom, M, et al., Nucleosides and Nucleotides 9: 123-7 (1990). Nick translation is frequently used as the enzymatic technique of choice for adding biotin to an oligonucleotide (Langer et al., Enzymatic synthesis of biotinylated polynucleotides: Novel nucleic acid affinity probes, Proc. Natl. Acad. Sci. U.S.A. 78:6633-6637 (1981).
Lately, biotin-phosphoramidite monomers have become available to take advantage of biotinylating in situ when performing automated polynucleotide synthesis. Edge, European Patent Application No. 88307934.5, discloses processes for automated synthesis of oligonucleotides in a DNA synthesizer. The polynucleotides so prepared may be used as probes for diagnostic tools in clinical diagnosis and research use. The biotin label is attached to the phosphoramidite monomer, which is then added to the growing polynucleotide chain. Similar biotin-phosphoramidite products are made by Amersham and Clontech. Recently, R. T. Pon disclosed an alternative method of making a long-chain phosphoramidite reagent for the automated synthesis of 5'-biotinylated oligonucleotides in Tetrahedron Letters 32(14): 1715-18 (1991). An N-dimethoxytritylated biotin group coupled to a 6-aminohexanol linker was converted into a phosphoramidite to yield a biotinylating reagent which can be used in automated solid phase synthesis to produce 5'-biotinylated oligonucleotides in high yields. However, in all of these biotin-phosphoramidites, the polynucleotide probe may not bind to its target sequence as well as an un-labelled probe due to the fact that the spacer-Biotin moiety now takes up the place of the omitted base, and so no base-pairing will occur where the biotin label is, affecting the hydridization characteristics. In cases where multiple labels are desired, this may be a significant drawback. In addition, some of these reagents have inadequately sized linker arms, or are insoluble in acetonitrile, a significant drawback.
Other biotinylating reagents (N.sup.4 -biotinylated deoxycytidine reagents) have been reported that can introduce biotin at any position. However, these reagents require lengthy and expensive synthetic procedures. See Roget, A., et al., Nucleic Acids Res. 17:7643-51 (1989); Pieles, U., et al., Nucleic Acids Res. 18:4355-60 (1990).
The use of 1-Phenylpyrazolin-5-one ("XPP") for activation of amino acid esters for solid phase peptide synthesis has been recently disclosed in U.S. Pat. No. 5,233,044 (Hudson). The '044 patent discloses the use of the enol amino acid esters of XPP for the efficient coupling of amino acids in solid-phase peptide synthesis ("SPPS"). The solid phase synthesis of peptides is a complex process which involves stepwise additions of amino-terminus-blocked amino acids to a peptide chain, the initial carboxyl terminus of which is attached to a solid support. SPPS typically begins with covalent attachment of the carboxyl end of a first alpha-amine protected amino acid through an organic linker to an insoluble resin synthesis bead. The general synthesis cycle then consists of deprotection of the alpha-amine group of the last amino acid, washing and, if necessary, neutralization, followed by reaction with a carboxyl-activated form of the next alpha-amine protected amino acid to be added. Each successive amino acid is attached to the terminal nitrogen by the carbonyl carbon of the carboxylic acid group. The '044 patent provides a unique reagent for activating amino acids for N-terminal addition, but does not disclose any method of labeling the resulting peptide.
There exists a need for more efficient and effective methods of adding non-radioactive marker molecules such as biotin and fluorescein to amine-functionalized biomolecules.